The present invention relates generally to a clinical chemical analyzing technique, and more particularly to an absorptiometric method of measuring concentrations and/or activities of chemical substances in sample liquids, in an accurate manner with a wide measurement range by measuring variations of absorbance with respect to time.
Such a method of measuring the concentrations and activities of substances by detecting variations of absorbance or optical density is sometimes called as kinetic assay and has been used for measuring an initial rate of reaction. In general, a reaction is carried out by combining a sample liquid with a reagent. Proceeding of reaction depends upon respective test items. FIG. 1 shows a typical curve representing a variation of absorbance with respect to time. The reaction process comprises a lag phase (a) in which the reaction proceeds slowly, a linear phase (b) in which the reaction proceeds linearly and an endpoint phase (c) in which the reaction is completed. In order to improve an accuracy of measurement of kinetic assay, it is absolutely necessary to effect measurement in the linear phase (b). For this purpose, various methods have been proposed. For instance, in one of known methods a measurement start point and a measurement end point have been previously determined for respective test items, and a measurement period defined by these points is divided into two halves. Values measured in these two halves are compared with each other to determine whether or not the measurement has been carried out in the linear phase (b). When the measurement is judged to be out of the linear phase (b), any analytical result is not calculated and an abnormal mark is printed out. However, in this known method, when the abnormal mark is printed out for a certain sample, the measurement has to be carried out again for the relevant sample and thus, an analyzing efficiency will be decreased. In order to increase the efficiency to effect accurate measurements in a short time period, it is necessary to increase the number of measurements for respective samples. That is to say, the number of samplings for measuring the absorbance change has to be increased to get a great amount of data and the data has to be processed statistically. Further, an admission of the lineality is judged on the basis of the following inequality. ##EQU1## wherein A and B are values measured in the front and back half periods, respectively. When the effectiveness of measurements is checked only by such an inequality, an influence of the lag phase could not be removed unless strict conditions would be imparted. If the condition is too strict, the abnormal mark might be printed out unnecessarily, even if variations are quite small.
In a Japanese Patent Application Laid-Open Publication No. 113,383/79 there is described another known absorptiometric method for measuring chemical substances. In this method, absorbances of respective test liquids are measured at three different time points. At first a variation in absorbance between first two measuring points is derived and is then compared with a standard value of variation. When the measured variation is larger than the standard value, i.e. when the sample contains given substance by a sufficiently large amount or when the activity of the sample is sufficiently high, the concentration or the activity of the given substance is calculated from the measured variation of absorbance. On the other hand, when the concentration or activity of the substance to be measured is low, the concentration or activity is calculated from a variation of absorbance derived from the measured absorbance values at the first and third measuring points. In this method since the concentration or activity is simply calculated from a difference between the absorbances measured at the two points in case of the high concentration, an accurate measurement could not be expected. In order to increase the measuring accuracy, it is preferable to gather data measured at several points and to use only data in the linear region, even in case of high concentration. Further, according to the known method the measuring range is only widened in case of low concentration and the reaction is not actually monitored during said range and thus, the reaction process could not be known and a high reliability could not be obtained. Moreover the absorbance measured at the first measuring point is always used. However, in particular, in case of enzyme catalyzed reaction measurement, the lag phase (a) is relatively long and the value measured at the first point is liable to be subjected to the influence of lag phase. Therefore, the measurement could not be carried out accurately.
In known methods, when the amount of a given substance of a sample to be measured is very small, which results in a very small variation in absorbance, the sample is usually treated as "measurement impossible". In this manner the ability of analysis is limited to a great extent. Further, if the absorbance per se or the variation in absorbance is abnormally high, the data is wasted as "abnormal data". Even in such a case it is preferable to derive analytic results as accurate as possible. However, if the analytic result thus obtained is derived as it is, it could not be distinguished from analytic results which are obtained by normal treatment. This is sometimes undesirable.